A tale of timing and transport

Excitatory and inhibitory synapses show long-term plasticity, but spike timing-dependent plasticity was seen only at excitatory connections. No longer. In this issue of Neuron, Woodin et al. demonstrate that coincident pre- and postsynaptic activity acts on the neuronal K+/Cl- cotransporter KCC2 to shift the reversal potential for Cl- and thus alters the effectiveness of GABAergic synapses.     

Immunolocalization of the acid-sensing ion channel 2a in the rat cerebellum

The acid-sensing ion channels (ASICs) are members of the DEG/ENaC superfamily of Na+ channels. Acid-gated cation currents have been detected in neurons from multiple regions of the brain including the cerebellum, but little is known about their molecular identity and function. Recently, one of ASICs (ASIC1a) was implicated in synaptic plasticity. In this study we examined the subcellular distribution of ASIC2a in rat cerebellum by immunostaining and confocal microscopy. Monoclonal antibodies for labeling of defined brain structures, for example, astroglia, Purkinje cell dendrites, nuclei, and presynaptic terminals were used for colocalization analyses. In the gray matter, the anti-ASIC2a antibody intensively stained dendrite branches of Purkinje cells evenly distributed throughout the entire molecular layer (ML). In the granule cell layer (GL), anti-ASIC2a antibody stained synaptic glomeruli. Neuronal localization of ASIC2a was confirmed by lack of co-staining with glial fibrillary acidic protein. Anti-ASIC2a staining in the ML colocalized with metabotropic glutamate receptor 1alpha (mGluR1alpha) in Purkinje cell dendrites and dendritic spines. Both proteins, mGluR1alpha and ASIC2a, were enriched in a crude synaptic membrane fraction prepared from cerebellum, suggesting synaptic expression of these proteins. Dual staining with anti-syntaxin 1A and anti-ASIC2a antibodies demonstrates characteristic complementary distribution of two proteins in both ML and GL. Because syntaxin 1A localized in presynaptic membranes and synaptic vesicles, complementary distribution with ASIC2a suggests postsynaptic localization of ASIC2a in these structures. This study shows specific localization of ASIC2a in both Purkinje and granule cell dendrites of the cerebellum and enrichment of ASIC2a in a crude cerebellar synaptic membrane fraction. The study is the first report of synaptic localization of ASIC2a in the CNS. The synaptic localization of ASIC2a in the cerebellum makes this channel a candidate for a role in motor coordination and learning.     

Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca²⁺-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.     

BDNF-induced TrkB activation down-regulates the K+-Cl- cotransporter KCC2 and impairs neuronal Cl- extrusion

Pathophysiological activity and various kinds of traumatic insults are known to have deleterious long-term effects on neuronal Cl- regulation, which can lead to a suppression of fast postsynaptic GABAergic responses. Brain-derived neurotrophic factor (BDNF) increases neuronal excitability through a conjunction of mechanisms that include regulation of the efficacy of GABAergic transmission. Here, we show that exposure of rat hippocampal slice cultures and acute slices to exogenous BDNF or neurotrophin-4 produces a TrkB-mediated fall in the neuron-specific K+-Cl- cotransporter KCC2 mRNA and protein, as well as a consequent impairment in neuronal Cl- extrusion capacity. After kindling-induced seizures in vivo, the expression of KCC2 is down-regulated in the mouse hippocampus with a spatiotemporal profile complementary to the up-regulation of TrkB and BDNF. The present data demonstrate a novel mechanism whereby BDNF/TrkB signaling suppresses chloride-dependent fast GABAergic inhibition, which most likely contributes to the well-known role of TrkB-activated signaling cascades in the induction and establishment of epileptic activity.     

2-Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice

Abstract: The 2-deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2-deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2-deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2-deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.     

A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons

We constructed a novel optical indicator for chloride ions by fusing the chloride-sensitive yellow fluorescent protein with the chloride-insensitive cyan fluorescent protein. The ratio of FRET-dependent emission of these fluorophores varied in proportion to the concentration of Cl and was used to measure intracellular chloride concentration ([Cl-]i) in cultured hippocampal neurons. [Cl-]i decreased during neuronal development, consistent with the shift from excitation to inhibition during maturation of GABAergic synapses. Focal activation of GABAA receptors caused large changes in [Cl-]i that could underlie use-dependent depression of GABA-dependent synaptic transmission. GABA-induced changes in somatic [Cl-]i spread into dendrites, suggesting that [Cl-]i can signal the location of synaptic activity. This genetically encoded indicator will permit new approaches ranging from high-throughput drug screening to direct recordings of synaptic Cl- signals in vivo.     

Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain

Antisera to calmodulin, a Ca2%-dependent modulator protein, and a heat- labile calmodulin-binding protein have been used to localize these proteins in mouse caudate-putamen. The two proteins appear to be located at identical sites in this brain area. At the light microscopic level, calmodulin and calmodulin-binding protein are found within the cytoplasm and processes of large cells. At the electron microscopic level the proteins are associated with neuronal elements only, primarily at postsynaptic sites within neuronal somata and dendrites. Within the dendrites the immunocytochemical label is associated predominantly with the postsynaptic density and dendritic microtubules. These results are in accord with recent biochemical and immunihistochemical studies of calmodulin in brain and in dividing cells. Thus, calmodulin and the heat-labile calmodulin-binding protein may play a role in the nervous system at the site of neurotransmitter action and at the level of microtubular function.     

Developmental regulation of the neuronal-specific isoform of K-Cl cotransporter KCC2 in postnatal rat brains.

We examined the expression of the KCC2 isoform of the K-Cl cotransporter in the developing and adult brain, using an affinity-purified antibody directed against a unique region of the KCC2 protein. Expression was shown to be limited to neurons at the cell bodies and cell processes in the hippocampus and cerebellum. Expression seemed to be the highest at the end of processes that originated from the CA1 pyramidal cells. Developmental up-regulation of KCC2 expression was demonstrated in the entire rat brain by Northern and Western blot analyses, and in the hippocampus by immunofluorescence. Level of KCC2 expression was minimal at birth and increased significantly during postnatal development. This pattern of expression was opposite to the one of the Na-K-2Cl cotransporter that is highly expressed in immature brain and decreases during development. The up-regulation of the K-Cl cotransporter expression is consistent with the developmental down-regulation of the intracellular Cl- concentration in neurons. The level of intracellular Cl-, in turn, determines the excitatory versus inhibitory response of the neurotransmitter gamma-aminobutyric acid in the immature versus mature brain. Finally, KCC2 expression was shown in dorsal root ganglion neurons, demonstrating that expression of the cotransporter is not strictly confined to central nervous system neurons.     

Dying-back of Purkinje cell dendrites with synapse loss in aging rats

Qualitative and quantitative changes were found in the cerebellar circuitry of old as compared to young rats. The old group had a reduced number of synapses (at least 30%), however, there was an increase in the size of remaining synaptic components (13.5% for spine head volume, 66% for bouton volume, and 17% for the area of synaptic contact zones). Furthermore, there were pronounced morphological changes in the older group appearing as: 1) prominent lipofuscin bodies in Purkinje cell somata, 2) numerous myelinated fibers in the lower part of the molecular layer, 3) tortuous Purkinje cell dendrites in a thinned molecular layer, and 4) abundant vacuolar profiles and membrane swirls in small and intermediate-sized dendrites. Our findings suggest that Purkinje cell dendrites are dying-back reducing the target field for granule cells and that remaining synaptic sites compensate by increasing synaptic contact area as well as the size of pre- and postsynaptic structures.     

Localization of five somatostatin receptors in the rat central nervous system using subtype-specific antibodies.

The cloning of five members of the somatostatin receptor family, sst1-sst5, as well as two isoforms of the somatostatin receptor 2, sst2A and sst2B, enabled us to generate specific anti-peptide antisera against unique sequences in the carboxyl-terminal tail of each somatostatin receptor subtype. We used these antibodies in multicolor immunofluorescent studies aimed to examine the regional and subcellular distribution of somatostatin receptors in adult rat brain. Several findings are notable: The cloned sst1 receptor is primarily localized to axons, and therefore most likely functions in a presynaptic manner. The cloned sst2 receptor isoforms exhibit strikingly different distributions, however, both sst2A and sst2B are confined to the plasma membrane of neuronal somata and dendrites, and therefore most likely function in a postsynaptic manner. The cloned sst3 receptor appears to be excluded from 'classical' pre- or postsynaptic sites but is selectively targeted to neuronal cilia. The cloned sst4 receptor is preferentially distributed to distal dendrites, and therefore most likely functions postsynaptically. The cloned sst5 receptor was not detectable in the adult rat brain, however, prominent sst5 expression was found in the pituitary. Furthermore, sst1-containing axons either co-contained somatostatin or were closely apposed by somatostatin-positive terminals in a regional-specific manner. Neuronal somata and dendrites containing either sst2A, sst2B or sst4 were found to exist in close proximity, although not necessarily synaptically linked, to somatostatin-positive terminals. Together, in the central nervous system the effects of somatostatin are mediated by several different receptor proteins which are distributed with considerable regional overlap. However, there appears to be a high degree of specialization among somatostatin receptor subtypes with regard to their subcellular targeting. This subtype-selective targeting may be the underlying principal of organization that allows somatostatinergic modulation of neuronal activity via both pre- and postsynaptic mechanisms.     

Electron microscopic localization of neuron-specific enolase in rat and mouse brain

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.     

GABA- and glycine-immunoreactive synapses in spinal cord of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Using the method of the double immune label combined with two antibodies, i.e., monoclonal antibodies to gamma-aminobutyric acid (GABA) and polyclonal antibodies to glycine, the distribution of gamma-aminobutyric acid- and glycine-immunoreactive synapses on motoneurons and primary afferent axons was studied in the frog Rana temporaria spinal cord. An analysis of all labeled boutons on the dendrites and soma of motoneurons showed the existence of three categories of immunoreactive synapses as follows: 7% were labeled for GABA, 23% were labeled for glycine, and approximately 70% were immunoreactive to both GABA and glycine. These results confirm the predominant role of glycine in the postsynaptic inhibition of motoneuronal activity. Three similar populations of synaptic boutons were also founded on primary afferent axons, including one GABA-immunoreactive (25%) and one glycine-immunoreactive (5%); the majority of the immunoreactive synapses had the colocalization of two inhibitory transmitters. As a rule, the higher proportion of axo-axonal synapses was organized in synaptic triads. The possible simultaneous roles of glycine as a transmitter of postsynaptic inhibition and as a transmitter that mediates the process of the autoreception of glutamate in the axo-axonal synapses on the primary afferent fibers are discussed.     

Immunohistochemical Localization of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Brain and Various Tissues

Polyclonal antibodies against Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen-affinity column. The antibodies which recognized both subunits of the enzyme with Mrs 49K and 60K were used for the study on the distribution of CaM kinase II in formalin-fixed, paraffin-embedded tissues. In the brain, a light-microscopic study demonstrated strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in nuclei. The densely stained regions included cerebral cortex, hippocampal formation, striatum, substantia nigra, and cerebellar cortex. In substantia nigra, neurites were stained, but not neuronal somata. Electron microscopy revealed that the immunoreactive product was highly concentrated at the postsynaptic densities. In addition to neurons, weak immunoreactivity was also demonstrated in glial cells, such as astrocytes and ependymal cells of ventricles and epithelial cells of choroid plexus. In other tissues, strong immunoreactivity was observed in the islet of pancreas and moderate immunoreactivity in skeletal muscle and kidney tubules. Immunoreactivity was demonstrated in all of the tissues tested. The results suggest that CaM kinase II is widely distributed in the tissues.     

Electroneutral Cation-Chloride Cotransporters in the Central Nervous System

Several members of the cation-chloride cotransporter (solute carrier family 12, SLC12) gene family are expressed within the central nervous system, with one family member, the K+-Cl- cotransporter KCC2, exclusive to neurons. These transporters are best known for their roles in cell volume regulation and epithelial salt transport, but are increasingly receiving attention in neuroscience. In particular, intracellular chloride activity and hence the neuronal response to GABA and glycine appears to be determined by a balance between chloride efflux and influx through KCC2 and the Na+-K+-2Cl- cotransporter NKCC1, respectively. This relationship has important implications for neuronal development, sensory perception, neuronal excitability, and the response to neuronal injury. Finally, the association between loss of function in the K+-Cl- cotransporter KCC3, with a severe peripheral neuropathy associated with agenesis of the corpus callosum, has revealed an unexpected role for K+-Cl- cotransport in the development and/or maintenance of both the central and peripheral nervous systems.     

Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture

Dissociated cultures from many species have been important tools for exploring factors that regulate structure and function of central neuronal synapses. We have previously shown that cells harvested from brains of late stage Drosophila pupae can regenerate their processes in vitro. Electrophysiological recordings demonstrate the formation of functional synaptic connections as early as 3 days in vitro (DIV), but no information about synapse structure is available. Here, we report that antibodies against pre-synaptic proteins Synapsin and Bruchpilot result in punctate staining of regenerating neurites. Puncta density increases as neuritic plexuses develop over the first 4 DIV. Electron microscopy reveals that closely apposed neurites can form chemical synapses with both pre- and postsynaptic specializations characteristic of many inter-neuronal synapses in the adult brain. Chemical synapses in culture are restricted to neuritic processes and some neurite pairs form reciprocal synapses. GABAergic synapses have a significantly higher percentage of clear core versus granular vesicles than non-GABA synapses. Gap junction profiles, some adjacent to chemical synapses, suggest that neurons in culture can form purely electrical as well as mixed synapses, as they do in the brain. However, unlike adult brain, gap junctions in culture form between neuronal somata as well as neurites, suggesting soma ensheathing glia, largely absent in culture, regulate gap junction location in vivo. Thus pupal brain cultures, which support formation of interneuronal synapses with structural features similar to synapses in adult brain, are a useful model system for identifying intrinsic and extrinsic regulators of central synapse structure as well as function.     

Modulation of GABAergic transmission by activity via postsynaptic Ca2+-dependent regulation of KCC2 function

Activity-induced modification of GABAergic transmission contributes to the plasticity of neural circuits. In the present work we found that prolonged postsynaptic spiking of hippocampal neurons led to a shift in the reversal potential of GABA-induced Cl- currents (E(Cl)) toward positive levels in a duration- and frequency-dependent manner. This effect was abolished by blocking cytosolic Ca2+ elevation and mimicked by releasing Ca2+ from internal stores. Activity- and Ca2+-induced E(Cl) shifts were larger in mature neurons, which express the K-Cl cotransporter KCC2 at high levels, and inhibition of KCC2 occluded the shifts. Overexpression of KCC2 in young cultured neurons, which express lower levels of KCC2 and have a more positive E(Cl), resulted in hyperpolarized E(Cl) similar to that of mature cells. Importantly, these young KCC2-expressing neurons became responsive to neuronal spiking and Ca2+ elevation by showing positive E(Cl) shifts. Thus, repetitive postsynaptic spiking reduces the inhibitory action of GABA through a Ca2+-dependent downregulation of KCC2 function.     

Immunohistochemical Localization of α-Mannosidase During Postnatal Development of the Rat Cerebellum

The localization of alpha-D-mannosidase in the rat cerebellum was studied by using indirect immunohistochemistry at both optical and electron microscopic levels. In the adult the enzyme is particularly concentrated in the dendrites and cell bodies of Purkinje cells, basket cells, and Golgi neurons in the cerebellar cortex and in the cytoplasm and dendrites of deep nuclei neurons. The cytoplasm of granule cells is poorly stained, whereas parallel fibers, white matter, Bergman fibers, and Golgi epitheloid cell perikarya show virtually no staining. Electron microscopy suggests that most of the staining is found in the cytosol, although some staining is found in the postsynaptic densities of the synapses between parallel fibers and Purkinje dendrites. The pattern of staining was followed throughout the postnatal development of the rat cerebellum. At bith an intense and diffuse staining is found in all cells except those of the external germinative layer. At the 6th postnatal day, Purkinje cell bodies and apical cones are strongly labeled. From the 13th day on the pattern is very similar to that found in the adult. However, at the 18th postnatal day (when compared with the other structures), the staining of Purkinje cell dendrites seems to be higher than at all other ages. These data are correlated with biochemical studies and discussed in relation to the possible role of this enzyme during the postnatal development of the rat cerebellum.     

Subcellular segregation of two A-type K+ channel proteins in rat central neurons.

In the mammalian nervous system, K+ channels regulate diverse aspects of neuronal function and are encoded by a large set of K+ channel genes. The roles of different K+ channel proteins could be dictated by their localization to specific subcellular domains. We report that two K+ channel polypeptides, Kv1.4 and Kv4.2, which form transient (A-type) K+ channels when expressed in Xenopus oocytes, are segregated in rat central neurons. Kv1.4 protein is targeted to axons and possibly terminals, while Kv4.2 is concentrated in dendrites and somata. This differential distribution implies distinct roles for these channel proteins in vivo. Their localizations suggest that Kv1.4 and Kv4.2 may regulate synaptic transmission via presynaptic, or postsynaptic mechanisms, respectively.